Piston-driven automated liquid handlers.

Laboratory capacities are often limited by time-consuming manual repetitive procedures rather than analysis time itself. While modern instruments are typically equipped with an autosampler, sample preparation often follows manual procedures including many labor-intensive, monotonous tasks. Particularly, for a high number of samples, well plates, and low microliter pipetting, manual preparation is error-prone often requiring repeated experiments. Sampling and sample preparation can account for greater analytical variability than instrument analysis. Repetitive tasks such as liquid handling benefit strongly from technological advances and led to the increasing applications of various automated liquid handlers (ALHs). In this review, we discuss the considerations for ALHs in the microliter range and highlight advantages and challenges when transforming from manual to automated workflows. We strongly focused on differences in liquid handling and outlined advantages due to sensor-controlled pipetting. ALHs can substantially improve costs-effectiveness and laboratory capacity. This is a consequence of increased efficiency, and throughput of laboratories while simultaneously raising data quality. Additionally, ALHs can improve safety, documentation of data, and sustainability. While automation requires careful consideration and resource demanding implementation, we believe it offers numerous advantages and can help to transform modern laboratories.

Protein Stability After Administration: A Physiologic Consideration.

Regulatory authorities and the scientific community have identified the need to monitor the in vivo stability of therapeutic proteins (TPs). Due to the unique physiologic conditions in patients, the stability of TPs after administration can deviate largely from their stability under drug product (DP) conditions. TPs can degrade at substantial rates once immersed in the in vivo milieu. Changes in protein stability upon administration to patients are critical as they can have implications on patient safety and clinical effectiveness of DPs. Physiologic conditions are challenging to simulate and require dedicated in vitro models for specific routes of administration. Advancements of in vitro models enable to simulate the exposure to physiologic conditions prior to resource demanding pre-clinical and clinical studies. This enables to evaluate the in vivo stability and thus may allow to improve the safety/efficacy profile of DPs. While in vitro-in vivo correlations are challenging, benchmarking DP candidates enables to identify liabilities and optimize molecules. The in vivo stability should be an integral part of holistic stability assessments during early development. Such assessments can accelerate development timelines and lead to more stable DPs for patients.

A Multicompany Survey Study for Helium Leak Container Closure Integrity Test.

All products labeled as sterile are required to be free of microbial contamination throughout their shelf life (obligatory critical quality attribute). Container closure integrity (CCI) needs to be addressed with a holistic life cycle strategy comprising adequate primary packaging components selection and the assessment of critical unit operations and critical process parameters (CPPs) according to quality by design (QbD) principles. The helium leak method is currently the most sensitive CCI test method and preferably used for the initial container closure system (CCS) qualification and characterization studies. Currently, two different measuring principles are used in the pharmaceutical industry, and no data is available in the public domain for typical method performance parameters such as accuracy, precision, intermediate-precision, and limit of quantification of the method. Furthermore, the performance of different types and sizes of artificial leaks as well as certified helium leak standards have not yet been characterized across different test laboratories. In this multicompany study, we shared 17 artificially prepared leak samples using the most common types of artificial leaks in relevant nominal size ranges that are commercially available or can be easily prepared in a laboratory. Each participating company generated results according to their in-house methods, applying their established test parameters as the aim of the study was not to create a standard for helium leak measurements, but to compare real-world performance between different laboratories. Consequently, this study is not an interlaboratory study using the same test method across laboratories.

Fate of antibody and polysorbate particles in a human serum model.

Monoclonal antibodies (mAbs) and excipients can degrade owing to different stress factors they encounter during their life cycle or after administration in human body. This can result in the formation of aggregates and particulates. As particles can evoke an immune response in patients, it becomes increasingly important to monitor their fate after administration. In this study, we used a protein-free serum model to assess the fate of mAb and polysorbate (PS) particles under physiologic conditions. Commonly encountered stress conditions such as pH, temperature, extrusion, and shaking were chosen to generate mAb particles. Alkaline hydrolysis was used to generate PS particles. The fate of aggregates and particles was evaluated in serum and histidine buffer. We observed that depending on the nature of stress and the environment particles are subjected to, the fate of particles can differ substantially. The mAb aggregates generated by pH stress, showed reduction in HMWS from 26% to 6% over 14 days in human serum filtrate. PS particles dissolved at 37 °C but remained unaltered in Histidine at 5 °C. Our results reinforce the need to track the fate of particles generated during drug product development upon exposure to physiologic conditions.

Assessment of Antibody Stability in a Novel Protein-Free Serum Model.

Therapeutic proteins can degrade upon administration as they are subjected to a variety of stresses in human body compartments. In vivo degradation may cause undesirable pharmacokinetic/pharmacodynamic profiles. Pre-clinical in vitro models have gained scientific interest as they enable one to evaluate the in vivo stability of monoclonal antibodies (mAbs) and ultimately can improve patient safety. We used a novel approach by stripping serum of endogenous proteins, which interfere with analytical test methods. This enabled the direct analysis of the target protein without laborious sample work-up procedures. The developed model retained the osmolality, conductivity, temperature, and pH of serum. We compared the impact of human, bovine, and artificial serum to accelerated stability conditions in histidine buffer. Target mAbs were assessed in regard to visible and sub-visible particles, as well as protein aggregation and fragmentation. Both mAbs degraded to a higher extent under physiological conditions compared to accelerated stability conditions. No relevant stability differences between the tested mAbs were observed. Our results reinforced the importance of monitoring protein stability in biological fluids or fluids emulating these conditions closely. Models enabling analysis in fluids directly allow high throughput testing in early pre-clinical stages and help in selecting molecules with increased in vivo stability.

Analytical Challenges Assessing Protein Aggregation and Fragmentation Under Physiologic Conditions.

Therapeutic proteins are administered by injection or infusion. After administration, the physiologic environment in the desired body compartment - fluid or tissue - can impact protein stability and lead to changes in the safety and/or efficacy profile. For example, protein aggregation and fragmentation are critical quality attributes of the drug product and can occur after administration to patients. In this context, the in vivo stability of therapeutic proteins has gained increasing attention. However, in vivo protein aggregation and fragmentation are difficult to assess and have been rarely investigated. This mini-review summarizes analytical approaches to assess the stability of therapeutic proteins using simulated physiologic conditions. Furthermore, we discuss factors potentially causing in vivo protein aggregation, precipitation, and fragmentation in complex biological fluids. Different analytical approaches are evaluated with respect to their applicability and possible shortcomings when it comes to these degradation events in biological fluids. Tracking protein stability in biological fluids typically requires purifying or labeling the protein of interest to circumvent matrix interference of biological fluids. Improved analytical methods are strongly needed to gain knowledge on in vivo protein aggregation and fragmentation. In vitro models can support the selection of lead candidates and accelerate the pre-clinical development of therapeutic proteins.

Stability of monoclonal antibodies after simulated subcutaneous administration.

Changes in the environment from the drug product to the human physiology might lead to physical and/or chemical modifications of the protein drug, such as in vivo aggregation and fragmentation. Although subcutaneous (SC) injection is a common route of administration for therapeutic proteins, knowledge on in vivo stability in the SC tissue is limited. In this study, we developed a physiologic in vitro model simulating the SC environment in patients. We assessed the stability of two monoclonal antibodies (mAbs) in four different protein-free fluids under physiologic conditions. We monitored protein stability over two weeks using a range of analytical methods, in analogy to testing purposes of a drug product. Both mAbs showed an increase of protein aggregates, fragments, and acidic species. mAb1 was consistently more stable in this in vitro model than mAb2, highlighting the importance of comparing the stability of different mAbs under physiologic conditions. Throughout the study, both mAbs were substantially less stable in bicarbonate buffers as compared to phosphate-buffered saline. In summary, our developed model was able to differentiate stability between molecules. Bicarbonate buffers were more suitable compared to phosphate-buffered saline in regards to simulating the in vivo conditions and evaluating protein liabilities.

Assessing Particle Formation of Biotherapeutics in Biological Fluids.

The stability of therapeutic proteins can be impacted in vivo after administration, which may affect patient safety or treatment efficacy, or both. Stability testing of therapeutic proteins using models representing physiologic conditions may guide preclinical development strategy; however, to date only a few studies assessing the physical stability are available in the public domain. In this manuscript, the stability of seven fluorescently labeled monoclonal antibodies (mAbs) was evaluated in human serum and phosphate-buffered saline, two models often discussed to be representative of the situation in humans after intravenous administration. Subvisible particles were analyzed using light obscuration, flow imaging, and imaging flow cytometry. All methods showed that serum itself formed particles under in vitro conditions. Imaging flow cytometry demonstrated that mean particle size and counts of mAbs increased substantially in serum over five days; however, particle formation in phosphate-buffered saline was comparably low. Stability differences were observed across the mAbs evaluated, and imaging flow cytometry data indicated that fluorescently labeled mAbs primarily interacted with serum components. The results indicate that serum may be more suitable as in vitro model to simulate physiologic intravenous conditions in patients closely and evaluate the in vivo stability of therapeutic proteins. Fluorescence labeling and detection methods may be applied to differentiate particles containing therapeutic protein from high amounts of serum particles that form over time.

Role of Formulation Parameters on Intravitreal Dosing Accuracy Using 1 mL Hypodermic Syringes.

PURPOSE: Evaluation of product viscosity, density and aeration on the dose delivery and accuracy for intravitreal injections with commonly used commercially available hypodermic 1 mL syringes. METHODS: Six commercially available hypodermic 1 mL syringes with different specifications were used for the study. Syringes were filled with the test solutions with different densities and viscosities. Syringes were also subjected to shaking stress to introduce aeration in the test solutions in the presence of different surfactant concentrations with and without high antibody concentration. Target intravitreal volumes of 100 µL, 50 µL and 30 µL were tested to assess dosing accuracy in a controlled simulated administration setup using DIN ISO 11040-4 guidelines and Zwick/Roell Z010 TN instrument. RESULTS: With increasing product viscosity, higher volumes and hence doses were delivered especially for very low volumes like 50 µL and 30 µL. No impact of increasing product density was found on the delivered dose. The presence of surfactants or high protein concentration can lead to aeration, which also negatively affects the dose accuracy and precision. CONCLUSION: Formulation parameters like viscosity can have an impact on dose delivery using hypodermic syringes for intravitreal injections and on the resulting glide force.

Determining Maximum Allowable Rubber Stopper Displacement for Container Closure Integrity (CCI).

Sterile pharmaceuticals require they be developed and manufactured using suitable container closure systems to maintain sterility until product opening. Characterizing container closure integrity (CCI) in relation to rubber stopper displacement was controversially discussed during the Annex 1 revision process. An automated inspection system can reject units with displaced rubber stoppers, and the related acceptance criteria for such in-process testing can be established by adequate studies. In this manuscript, we describe a novel helium leak CCI testing method to study the relation of rubber stopper displacement and CCI. Ten different commonly used vial-rubber stopper combinations were characterized, which led to robust test results. Pronounced differences between the different vial-rubber stopper combinations were observed, clearly showing that the combination of different stoppers, vials, and caps led to significant differences in allowable stopper displacement for routine manufacture.

Characterization of Polymeric Syringes Used for Intravitreal Injection.

Intravitreal (IVT) injection is currently the state of the art for drug delivery to the back of the eye. Drug Products (DP) intended for IVT injections usually pose challenges such as a very low injection volume (e.g. 50 µL) and high injection forces. DPs in vials are typically transferred and injected using disposable polymer syringes, which can feature a silicone oil (SO) coating. In our syringe in-use study, we compared dead volume, total SO content and SO layer distributions of three IVT transfer injection syringes. We assessed multiple potential impact factors such as protein concentration, needle gauge, injection speed, surfactant type and the impact of the in-use hold time on sub-visible particle (SvP) formation and injection forces. Pronounced differences were observed between the syringes regarding SvP generation. Siliconized syringes showed higher SvP counts as compared to non-siliconized syringes. In some cases injection forces exceeded 20 N, which caused needles to burst off during injection. The syringes also showed relevant differences in total SO content and dead volume. In conclusion, specific consideration in the selection of an adequate transfer injection syringe are required. This includes extensive testing and characterization under intended and potential in-use conditions and the development of in-use handling procedures.

Tracking the physical stability of fluorescent-labeled mAbs under physiologic in vitro conditions in human serum and PBS.

In recent years, the stability of biotherapeutics in vivo has received increasing attention. Assessing the stability of biotherapeutics in serum may support the selection of adequate molecule candidates. In our study, we compared the physical stability of 8 different monoclonal antibodies (mAbs) in phosphate-buffered saline (PBS) and human serum. mAbs were Alexa Fluor 488-labeled and characterized with respect to fragmentation, aggregation, and proteinaceous particle formation. Samples were analyzed using size-exclusion chromatography, light obscuration, and flow imaging. In addition, novel methods such as flow cytometry and fluorescence microscopy were applied. mAbs were selected based on their hydrophobicity and isoelectric point. All mAbs studied were inherently less stable in human serum as compared to PBS. Particle size and particle counts increased in serum over time. Interestingly, certain mAbs showed significant levels of fragmentation in serum but not in PBS. We conclude that PBS cannot replicate the physical stability measured in serum. The stability of labeled mAbs in human serum did not correlate with their hydrophobicity and isoelectric point . Serum stability significantly differed amongst the tested mAbs.

Evaluation of Different Quality-Relevant Aspects of Closed System Transfer Devices (CSTDs).

PURPOSE: Health care professionals can be exposed to hazardous drugs such as cytostatics during preparation of drugs for administration. Closed sytem transfer devices (CSTDs) were introduced to provide protection for healthcare professional against unintended exposure to hazardous drugs. The interest in CSTDs has significantly increased after USP <800> monograph was issued. The majority of the studies published so far on CSTDs have focused on their "containment" function. However, other important attributes for CSTDs with potential importance for product quality impact are not yet fully evaluated. METHODS: In the current study, we evaluated four sytems from different suppliers, in combination with different container closure systems (CCS), using solutions of different viscosity and surface tension. The different CSTD / CCS combinations were tested for (a) containment (integrity) using a highly sensitive helium leak test, (b) the force required for mounting the vial adaptor, (c) contribution to visible and subvisible particles as well as (d) the hold-up volume. RESULTS: Results show that the majority of CSTDs may have leaks varying in size, and that some of them generated visible particles due to stopper coring and subvisible particles, both due to silicon oil and particulate contaminations of the Devices. Finally, the holdup volume was up to 1 mL depending on the CSTD type, vial size and solution viscosity. CONCLUSION: These results show that there is a need to evaluate the compatibility of CSTD systems to select the best system for the intended use and that CSTDs may adversely impact product quality and delivered dose.

Particle Analysis of Biotherapeutics in Human Serum Using Machine Learning.

In recent years, an increasing number of studies assessed the stability of biotherapeutics in biological fluids. Such studies aim to simulate the conditions encountered in the human body and investigate the in vivo stability under in vitro conditions. However, on account of complexity of biological fluids, standard pharmaceutical methods are poorly suited to assess the stability of biotherapeutics. In this study, a fluorescent-labeled therapeutic immunoglobulin G (IgG) was analyzed for proteinaceous particles after mixing with human serum and after incubation at 37°C for 5 days. Samples were analyzed using standard pharmaceutical methods (light obscuration and dynamic imaging). Moreover, we developed a fluorescence microscopy method allowing to semiquantitatively detect IgG particles in serum. Several hundred IgG particles were detected after exposure to serum. Moreover, particle counts and particle size increased in serum over time. The results showed that an IgG may form particles on mixing with serum and novel methods such as fluorescence microscopy are required to gain insight on the stability of biotherapeutics in biological fluids. Furthermore, we showed distinct advantages of machine learning over traditional threshold-based methods by analyzing microscopy images. Machine learning allowed simplifying particles in regards to count, size, and shape.

Correction to: In Vivo Stability of Therapeutic Proteins.

A manuscript version without peer-review revisions was mistakenly processed and published.

In Vivo Stability of Therapeutic Proteins.

Significant efforts are made to characterize molecular liabilities and degradation of the drug substance (DS) and drug product (DP) during various product life-cycle stages. The in vivo fate of a therapeutic protein is usually only considered in terms of pharmacokinetics (PKs) and pharmacodynamics (PDs). However, the environment in the human body differs substantially from that of the matrix (formulation) of the DP and may impact on the stability of an injected therapeutic protein. Stabilizing excipients used in protein formulations are expected to undergo more rapid distribution and dissociation in vivo, compared to a protein as a highly charged macromolecule. Thus, in vivo stability may significantly differ from shelf-life stability. In vivo degradation of the therapeutic protein may alter efficacy and/or safety characteristics such as immunogenicity. Studying the stability of a therapeutic protein in the intended body compartment can de-risk drug development in early stages of development by improving the selection of better clinical lead molecules. This review assesses the considerations when aiming to evaluate the in vivo fate of a therapeutic protein by comparing the physiology of relevant human body compartments and assessing their potential implications on the stability of a therapeutic protein. Moreover, we discuss the limitations of current experimental approaches mimicking physiologic conditions, depending on the desired route of administration, such as intravenous (IV), subcutaneous (SC), intravitreal (IVT), or intrathecal (IT) administration(s). New models more closely mimicking the relevant physiologic environment and updated analytical methods are required to understand the in vivo fate of therapeutic proteins.

Method to Predict Glass Vial Fogging in Lyophilized Drug Products.

Glass fogging is a phenomenon occurring in lyophilized drug products and can be described as a thin product layer deposited on the inner surface of the glass container, in the area not covered by the lyo cake itself. It is often considered a cosmetic defect; however, the loss of container closure integrity is a potential consequence of the fogging's expansion to the vial neck region, making this a potential critical defect. Thus, a method for predicting the extent of vial fogging before the actual freeze-drying is of particular interest for the pharmaceutical industry. For that reason, we evaluated a simple method ("simulated fogging") applicable to drug product formulations in a specific container closure system. Two different vial types with different surface hydrophilicity were tested using 3 model protein formulations, comparing the simulated fogging test and the degree of fogging after actual lyophilization. The simulated fogging method could predict fogging and showed a correlation to fogging in lyophilized drug product glass vials. We observed that all formulations showed fogging in the hydrophilic vials. By contrast, hydrophobic vials prevented fogging, however, interestingly with remaining defects of so-called droplet formation. Other than extent of fogging, no additional differences of lyophilized cake properties or other product quality attributes were observed between products using the different glass vial types tested.

Subvisible Particulate Contamination in Cell Therapy Products-Can We Distinguish?

Cell therapy products represent an exciting new class of medicinal products, which must be parenterally administered. Thus, compliance with parenteral preparation guidelines is required. One requirement for parenteral products is the characterization of particle contaminations. As cell-based products are turbid suspensions, containing particles, the cells, characterization and control of foreign particle impurities remain a challenge. Within this study, we evaluated a flow imaging microscopy method for the detection and characterization of subvisible particle contaminations in cell-based products. We found that flow imaging microscopy is a potential method where subvisible particle contaminations can be differentiated from the cells in cell therapy products.

Shape Characterization of Subvisible Particles Using Dynamic Imaging Analysis.

Protein aggregates and subvisible particles (SbvP), inherently present in all marketed protein drug products, have received increasing attention by health authorities. Dynamic imaging analysis was introduced to visualize SbvP and facilitate understanding of their origin. The educational United States Pharmacopeia chapter <1787> emphasizes that dynamic imaging analysis could be used for morphology measurements in the size range of 4-100 µm. However, adequate morphology characterization, as suggested in the United States Pharmacopeia <1787> proposed size range, remains challenging as nonspherical size standards are not commercially available. In this study, a homogenous and well-defined nonspherical particle standard was fabricated and used to investigate the capabilities of 2 dynamic imaging analysis systems (microflow imaging (MFI) and FlowCAM) to characterize SbvP shape in the size range of 2-10 µm. The actual aspect ratio of the SbvP was measured by scanning electron microscopy and compared to the results obtained by dynamic imaging analysis. The test procedure was used to assess the accuracy in determining the shape characteristics of the nonspherical particles. In general, dynamic imaging analysis showed decreasing accuracy in morphology characterization for 5 µm and 2 µm particles. The test procedure was also capable to compare and evaluate differences between the 2 dynamic imaging methods. The present study should help to define ranges of operation for dynamic imaging analysis systems.

Phase-Appropriate Application of Analytical Methods to Monitor Subvisible Particles Across the Biotherapeutic Drug Product Life Cycle.

The phase-appropriate application of analytical methods to characterize, monitor, and control particles is an important aspect of the development of safe and efficacious biotherapeutics. The AAPS Product Attribute and Biological Consequences (PABC) focus group (which has since transformed into an AAPS community) conducted a survey where participating labs rated their method of choice to analyze protein aggregation/particle formation during the different stages of the product life cycle. The survey confirmed that pharmacopeial methods and SEC are the primary methods currently applied in earlier phases of the development to ensure that a product entering clinical trials is safe and efficacious. In later phases, additional techniques are added including those for non-GMP extended characterization for product and process characterization. Finally, only robust, globally-accepted, and stability-indicating methods are used for GMP quality control purposes. This was also consistent with the feedback during a webinar hosted by the group to discuss the survey results. In this white paper, the team shares the results of the survey and provides guidance on selecting phase-appropriate analytical methods and developing a robust particle control strategy.